Congenital heart disease (CHD) may either cause heart failure in early development, or trigger heart dysfunction in adulthood. Adult heart disease affects ~ 5.1 million people in the United States, and is the leading cause of death for adults. Unlike many other cell types in the mammalian body, cardiomyocyte proliferation ceases shortly after birth. Thus, the adult mammalian heart processes a very limited regenerative capacity, if it exists at all. Significant cardiomyocyte loss occurs in cardiac disease, therefore it is critical t identify the molecules and pathways that regulate cardiomyocyte proliferation and growth. The Hippo signaling pathway is involved in regulating cell growth and organ size, mediated by the core components Mst1/2 and Lats1/2 kinases controlling the subcellular localization of the downstream effectors Yap/Taz. We have recently demonstrated that Yap is necessary and sufficient for cardiomyocyte proliferation, survival during embryogenesis and during neonatal or later stages in the mammalian heart. Overexpression of a constitutively active form of Yap leads to an increase in heart size and extends the cardiac regeneration window in neonatal mice. Despite these advances, the upstream regulatory proteins and the downstream targets of Yap/Taz that mediate cardiomyocyte proliferation are largely unknown. Recent studies in cancer cell lines have demonstrated that G-protein coupled receptor (GPCR) signaling is able to regulate the Hippo pathway via heterotrimeric G proteins, with different G alpha subunits having distinct functions. Our preliminary data suggest that G?s inhibits cardiomyocyte proliferation by inhibiting Yap nuclear localization in vivo, while Yap negatively regulates the expression of Gnas, gene coding for G?s, by binding to the promoter of Gnas. Based on these data, our central hypothesis is that G proteins and Yap regulatory circuitry is essential for cardiomyocyte growth. To test the hypothesis, we have the following specific aims: 1) to determine the requirement of G proteins in regulation of cardiomyocyte proliferation through modulation of Yap activity and 2) to define the molecular mechanism whereby Yap regulates cardiomyocyte growth. We will use new conditional knock-out mouse strains to selectively ablate Gnas in cardiomyocytes at different developmental stages. We will further employ whole genome strategies to identify direct target genes of Yap and Taz to elucidate the molecular mechanism of Yap/Taz-mediated regulation of cardiomyocyte proliferation and heart repair. The objective of this proposal is to 1) elucidate the fundamental molecular mechanism of crosstalk between the GPCR and Hippo signaling pathways during heart development, and 2) develop therapeutic strategies for heart repair by defining Hippo signaling effectors in heart development and regeneration. These studies will provide invaluable insight into the mechanisms of cardiomyocyte proliferation, heart regeneration, and pathogenesis.